Lysozyme is a mucopeptide glycohydrolase enzyme which hydrolyzes 1,4-B links between N-acetylmuramic acid and N-acetylglucosamine, and is thus destructive to cell walls of certain microbes. Commercially available lysozyme has been used in, for example, dentifrices, chewing gums, and contact lens cleaners.
Ruminant stomach lysozyme is a lysozyme characteristic of the stomach mucosa of mammals with foreguts. It is apparently not in wide commercial usage. Endo-.beta.-N-acetylglucosaminidases have generally been used as analytical tools for the structural study of carbohydrates and or glycoproteins. We have found that the antimicrobial effectiveness of ruminant stomach lysozyme plus endo-.beta.-N-acetylglucosaminidase and/or endoglycopeptidase is greater than the antimicrobial effectiveness of either enzyme alone.
The use of lysozyme in mixtures for use as antimicrobials has been noted in dental rinse (U.S. Pat. No. 4,355,022, Rabussay, issued Oct. 19, 1982). Rabussay discloses a method for removing plaque and calculus comprising applying a solution containing lysozyme (0.1 ml/mg), and optionally also applying lipases, phospholipases, carbohydrases, and/or proteases. A dental treatment agent comprising the mixture is also disclosed by Rabussay. Carbohydrase is a general class of enzymes to which endoglycosidase belongs.
Australian Patent 8548514, Neeser, May 1, 1986 discloses an antibacterial composition containing a glycopeptide (I) and/or oligosaccharide. In the preparation, a glycoprotein (A) of plant origin is digested with a proteolytic enzyme, then optionally the glycopeptide product is converted to oligosaccharides by treatment with endo-beta-N-acetylglucosaminidase-H. The product can then be digested with an exo-alpha-mannosidase to preferentially cleave the alpha 1-2 bonds between mannose residues. The isolated glycopeptide (I) is claimed to be effective against pathogenic bacteria having type I fimbriae (e.g. Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium or Shigella flexneri).
Japanese Patent 62 044 180, laid open Feb. 26, 1987 discloses an endo-.beta.-N-acetylglucosaminidase (I) which reacts with the N,N'-diacetylchitobiose structure of asparagine-bonded sugar of glycoprotein to hydrolyze the beta-4 bond of N-acetylglucosamine and isolate oligosaccharide from glycoprotein. It has substrate specificity such that (I) reacts with high mannose type or mixed type of asparagine bonded saccharide chain and with complexed products. The enzyme is obtained from Canavalia gladiata DC. It is useful for researching bioactivity of the sugar chain portions of glycoproteins. Enhancement of ruminant stomach lysozyme action through endoglycosidase action has not been described, however.
Three copending U.S. patent applications filed on the same day as this patent application, describe methods and formulations comprising Type II endoglycosidases, a group which includes the instant endoglycosidases. The first copending patent application, entitled "Method and Formulation Employing Type II Endoglycosidase" and whose inventors are R. S. Carpenter, A. M. Wolff, P. J. Lad, and I. J. Goldstein, describes Type II endoglycosidases for removal of glycoside-containing substances. The second copending patent application, entitled "Method Employing Type II Endoglycosidase", whose inventors are R. S. Carpenter, A. M. Wolff, and P. J. Lad, describes Type II endoglycosidases for removal of microorganisms. The third copending patent application, entitled "Antimicrobial Method and Formulation Employing Type II Endoglycosidase and Antimicrobial Agent", whose inventors are R. S. Carpenter, A. M. Wolff, and P. J. Lad, describes the combination of Type II endoglycosidases and antimicrobial agents. The present patent application describes an exemplary benefit gained from combining ruminant stomach lysozyme with endo-.beta.-N-acetylglucosaminidase and/or endoglycopeptidase.